Bronchodilators are used in the treatment of asthma for acute relief of bronchospasm and for long-term control. Currently, only one class of direct bronchodilators, p-agonists acting at airway smooth muscle P2- adrenergic receptors, are in use. With many asthmatics not achieving adequate disease control, there is a need for additional therapeutics, including direct bronchodilators acting by novel mechanisms. We have discovered multiple bitter taste receptor (TAS2R) subtypes on human airway smooth muscle (HASM). Activation of TAS2Rs by bitter ligands causes marked airway smooth muscle relaxation in vitro and in vivo, and in various models of asthma. TAS2R-mediated bronchodilation is as efficacious as p-agonists, and is via a completely different mechanism involving specialized Ca^^ signaling. There are thousands of potential TAS2R agonists derived from plants or synthesized for other purposes, yet prior to our findings these were not considered for asthma therapy. In this Project, we will examine the properties of HASM TAS2Rs with an eye towards agonists being a new class of bronchodilators for treating asthma. In Aim 1, we will focus on the potential for acute desensitization, which can limit therapeutic efficacy. Agonist-promoted desensitization of the 3 most abundant HASM TAS2Rs will be examined in HASM cells, bronchi from precision-cut human lung slices and recombinantly expressing model cells. G-protein coupling, phosphorylation, internalization, B arrestin interactions, and biased ligand properties will be established. The influence ofthe asthma diathesis on these properties will also be determined using 2 models, so that applicability to asthma treatment can be ascertained. In Aim 2, we will establish the specific GRK phosphorylation sites for the relevant HASM TAS2RS, using site-directed mutagenesis and recombinant expression, so that a precise mechanism ofthe signal quenching is determined. Some TAS2Rs appear to undergo upregulation when exposed chronically to agonists, a phenotype that is opposite to P2-adrenergic receptors which downregulate upon chronic p-agonist treatment. TAS2R upregulation would be an extremely favorable profile, acting to limit tachyphylaxis. Aim 3 will determine which ofthe HASM TAS2Rs display upregulation, the molecular mechanism ofthe process, and the basis for biased ligands to promote the effect. Collectively, these studies will define the molecular and pharmacologic properties of HASM TAS2Rs required for future development of a novel class of bronchodilators for treating asthma.